In biological and medical research, examining the formation of three-dimensional (3D) aggregate(s) of eukaryotic cells, such as tumor cell aggregate(s) and embryoid bodies (EBs), is important for evaluating various drug screenings, biological compatibility, and cell therapies as well as determining gene/protein expression profiles and engineering cell tissue for various purposes. In recent years, in order to alleviate unnecessary animal suffering, considerable effort has gone into growing eukaryotic cell aggregate(s) to simulate such cell growth in vivo.
Classically, in vitro eukaryotic cells have been cultured as a monolayer on two-dimensional (2D) surfaces made of synthetic materials, such as glass and plastic. However, it is generally accepted that in vitro 3D cell aggregate(s) are physiologically more relevant in comparison to 2D cell monolayer(s), and thus can provide a more accurate precursor model in simulating in vivo animal studies. In the last decade, various studies have demonstrated that in vitro 2D cell monolayers do not accurately represent the in vivo microenvironment, nutrient intake, or biochemical processes including protein production. In addition, the gene/protein expression profiles for a variety of cancer and stem cell lines are also very different in 2D and 3D models. In the past decade, many different in vitro methods of growing 3D cell aggregate(s) have been evaluated to accurately simulate in vivo models used in research studies. However, producing a product that facilitates the formation of 3D cell aggregate(s) has proven to be a difficult design challenge.
Aggregate or spheroid size is an important parameter for screening assays. Single, uniform-sized 3D cell aggregates are desirable for repeatability and accuracy in experiments. The generation of a single, uniform sized spheroid per well of a cell culture device allows screening and testing of biochemical compounds, biological agents and infectious organisms as well as allowing for toxicity evaluations. In the case of embryoid bodies (EBs), spheroid size is linked to cell differentiation potential. In widely used hydrophilic dishes for spheroid culture, agglomeration of spheroids is an area of concern. Large agglomerates can produce their own microenvironment, within which cells produce their own growth factors. This makes it harder to control the culture environment and determine the effects of exogenous factors. Therefore, to identify tumor spheroids or EBs as model systems, it is necessary to form them individually in a uniform and reproducible manner with regulated homogeneity in morphology and differentiation status. Indeed, for screening purposes, uniform sized EBs have been shown to have synchronous differentiation potential.
An in vitro technique widely used to grow eukaryotic cell 3D aggregate(s) is referred to as the Hanging Drop Method. This method has proven useful both for growing cancer cell lines into tumor spheroids and for growing embryonic stem cells into EBs. To practice this method, a cell culture medium (e.g., a sterile water solution containing nutrients), having a known concentration of cells, is added as drops to a flat horizontal surface such as glass or plastic (e.g., the underside of a glass Petri dish cover). Sterile water is then placed in the mating bottom portion (e.g., into the mating bottom part of a glass Petri dish) to maintain humidity within the dish. The dish cover with the suspended drops containing cells in culture medium is gently inverted and is placed atop the mating bottom portion. Due to gravity acting on the cell culture drops suspended from the inverted surface, the cells, being heavier than the medium, settle from the cell suspension in the medium to the bottom of the meniscus of each hanging drop. This unique configuration allows for only the top part of the medium (e.g., the part hanging from the Petri dish lid), which is cell-poor, to be in contact with substrate, and, as a result, the cells form 3D aggregate(s) in the cell medium and do not come in contact with any synthetic surface (e.g., glass or plastic) used in 2D monolayer techniques.
However, there are multiple drawbacks to the Hanging Drop Method. For example, the drops of medium are held on the surface of the Petri dish only by surface tension and adhesion forces, resulting in drop size being limited to a volume of 50 μL or less to resist the gravitational force pulling down on each drop. A further drawback to this method is that the drops are accessible only if the Petri dish lid containing the drops is gently inverted. Thus, it becomes difficult to conveniently change the medium surrounding the cells and to periodically observe the growth of the cell aggregate(s) using a microscope. Furthermore, the agitation caused by inverting the dish can easily cause the drops to run together or fall into the bottom half of the Petri dish. Additionally, if the plate is bumped or rocked even slightly, the hanging drops can easily fall into the media or sterile liquid below them, thus making the recovery of the cells/aggregate(s) contained in the drops highly problematic. On average, only 50-60% of aggregate(s) can be recovered using this method. Moreover, this method is inherently incapable of large-scale production.
In some culture devices, the hanging drop plate consists of a body with two coplanar surfaces having a narrow cylindrical- or hyperboloid-shaped communication conduit connecting the surfaces. The liquid drop is added to a widened portion of the inlet compartment located in the top plate and passes through the conduit to reach a circular relief structure located on the underside of the bottom plate that allows the pendant drop to adhere without spreading on the bottom plate. This design allows for convenient access for easily replenishing the liquid. However, the issue of gravitational instability of the pendant drops used to culture the 3D cell aggregate(s) remains unresolved. Additionally, an inverted microscope cannot be employed to observe the hanging drops.
Another culture system describes a method for producing biological organic material in a substantially spherical drop comprising a culture medium, the drop residing on a substrate in a non-adhered state, and the substrate having a water contact angle of at least 150°. In order to maintain the drop in a fixed state, a specially constructed cell culture plate having periodic protrusions to surround each spherical drop is required. The drops are non-adhered and thus can move between the protrusions; this movement may allow the drops to merge together resulting in the undesirable loss of single drops and spheroids. Furthermore, direct microscopic analysis of the cell aggregate(s) in the drop form is not possible without removing the drop and transferring it to a fresh surface or vessel.